Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

Most cancer cells rely on aerobic glycolysis to support uncontrolled proliferation and evade apoptosis. However, pancreatic cancer cells switch to glutamine metabolism to survive under hypoxic conditions. Activation of the Wnt/β-catenin pathway induces aerobic glycolysis by activating enzymes required for glucose metabolism and regulating the expression of glutamate transporter and glutamine synthetase. The results demonstrate that riluzole inhibits pancreatic cancer cell growth and has no effect on human pancreatic normal ductal epithelial cells. RNA-seq experiments identified the involvement of Wnt and metabolic pathways by riluzole. Inhibition of Wnt-β-catenin/TCF-LEF pathway by riluzole suppresses the expression of PDK, MCT1, cMyc, AXIN, and CyclinD1. Riluzole inhibits glucose transporter 2 expression, glucose uptake, lactate dehydrogenase A expression, and NAD + level. Furthermore, riluzole inhibits glutamate release and glutathione levels, and elevates reactive oxygen species. Riluzole disrupts mitochondrial homeostasis by inhibiting Bcl-2 and upregulating Bax expression, resulting in a drop of mitochondrial membrane potential. Finally, riluzole inhibits pancreatic cancer growth in KPC (Pdx1-Cre, LSL-Trp53R172H, and LSL-KrasG12D) mice. In conclusion, riluzole can inhibit pancreatic cancer growth by regulating glucose and glutamine metabolisms and can be used to treat pancreatic cancer.

Measurement of NAD + . Pancreatic cancer cells were seeded in 24-well plate and treated with riluzole (0-10 µM) for 1 h and total cellular NAD + concentration was measured at 450 nm by a NAD/NADH assay kit (Cayman).
Glutamate release was measured by the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit with a fluorometer using excitation at 540 nm and emission at 590 nm (Invitrogen).

Measurement of intracellular GSH. Pancreatic cancer cells and CSCs were treated with riluzole
(0-10 µM) for 24 h. Intracellular total GSH was detected by measuring the product of glutathionylated DTNB at 405 nm (GSH assay kit, Cayman Chemical).

Measurement of reactive oxygen species.
Cells were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0-10 µM) for 24 h. Cells were labeled with 2' ,7'-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured with a fluorometer using excitation at 495 nm and emission at 529 nm (Cellular Reactive Oxygen Species Detection Assay Kit, Abcam). Quantitative real-time PCR. Quantitative real-time PCR was performed as we described elsewhere 37 .
In brief, total RNA was first extracted with TRIzol reagent (Invitrogen), and the cDNA was generated by the Reverse Transcription System (Promega) in a 20 μl reaction containing 1 μg of total RNA. A 0.5 μl aliquot of cDNA was amplified by Fast SYBR Green PCR Master Mix (Applied Biosystems) in each 20 μl reaction. PCR reactions were run on the ABI 7900 Fast Real-Time PCR system (Applied Biosystems).
The following gene-specific primers were used: KPC (Pdx1-Cre, LSL-Trp53 R172H , and LSL-Kras G12D ) mice. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the LSU Health Sciences Centre, New Orleans. All experimental procedures also followed the relevant guidelines and regulations according to state, national and international standards for ethics in animal experimentation. Additionally, this study is compliant with the ARRIVE guidelines. KPC (Pdx1-Cre, LSL-Trp53 R172H , and LSL-Kras G12D ) mice were generated as described elsewhere 44 .
KPC mice (about 6 weeks old, n = 7) were injected ip with or without riluzole (20 mg/kg, Monday through Friday) for about 12 weeks 44 . At the end of the treatment, mice were euthanized by CO2 inhalation followed by thoracotomy. Histological examination of the pancreas was performed by H&E staining. Numbers of PanINs and PDAC were quantified as described elsewhere 45 . Animals were kept in pathogen-free conditions, at 21 to 25 °C and exposed to a normal diurnal variation under 12 h of light and 12 h of dark with food and water available ad libitum. Mice were weighed and observed regularly for signs of distress.
Quantification and statistical analysis. GSEA v2.1.0 GraphPad PRISM 7, R 3.2.3 and Python 2.7.2 software packages were used to perform the statistical analyses. Statistical differences between groups were analyzed using the Student t test or Analysis of Variance (ANOVA). The threshold for statistical significance is p < 0.05, unless otherwise stated. The mean ± SD or SEM was calculated for each experimental group, unless otherwise specified.

Results
Riluzole inhibited cell viability and colony formation, and induced apoptosis in pancreatic cancer cell lines and CSCs while sparing human normal pancreatic ductal epithelial cells. We first examined the effects of riluzole on pancreatic cancer cells and CSCs. Pancreatic CSCs (CD133 + , CD24 + , CD44 + , and ESA + ) were isolated from primary tumors and characterized as we described earlier 46 . Riluzole inhibited cell viability and colony formation in pancreatic cancer cell lines (AsPC-1, PANC-1, MIA PaCa-2) and CSCs ( Fig. 1  A). By comparison, riluzole had no effect on the viability of human pancreatic normal ductal epithelial (HPNE) cells (Fig. 1B). Riluzole inhibited colony formation in pancreatic cancer cell lines and CSCs (Fig. 1C). Riluzole induced apoptosis in pancreatic cancer cell lines and CSCs (Fig. 1D). Maximum induction of apoptosis was seen in PANC-1 cells, whereas Pan CSCs were least responsive. These data suggest that riluzole can be used for the treatment of pancreatic cancer.
Riluzole regulated Wnt/β-catenin/TCF-LEF pathway and mitochondrial proteins. We next perform RNA-Seq experiment to identify pathways which regulate cell metabolism and mitochondrial functions in response to treatment with riluzole. As shown in Fig. 2A, gene expression pattern was different in control and riluzole-treated cells. Riluzole mainly regulated Wnt/β-catenin/TCF-LEF, MAP kinase, TNF and mitochondrial pathways (Fig. 2B). Since our focus was to examine the effects of riluzole on cancer cell metabolism, we selected to examine the regulation of Wnt/β-catenin/TCF-LEF pathway which controls the expression of several genes required for glucose and glutamine metabolisms, and mitochondrial function.
Riluzole inhibited components of β-catenin/TCF-LEF pathway. Since our RNA-Seq experiments identified the regulation of β-catenin/TCF-LEF1 pathway by riluzole in pancreatic cancer, we examined the effects of riluzole on the expression of components of this pathway by q-RT-PCR and TCF-LEF1 transcriptional activity by luciferase assay (Fig. 3). Riluzole inhibited the expression of β-catenin, Wnt3a, Wnt5a, WSP, TCF and LEF in both MIA PaCa-2 and AsPC-1 pancreatic cancer cells (Fig. 3A-F and I-N). Since riluzole inhibited the expression of genes in β-catenin pathway, we next sought to measure the TCF-LEF1 transcriptional activity which is regulated by β-catenin. Riluzole inhibited TCF-LEF1 transcriptional activity in both MIA PaCa-2  Riluzole inhibited the expression of TCF-LEF1 target genes. The Wnt/β-catenin pathway is dysregulated in pancreatic ductal adenocarcinoma 47,48 . Although the activation of this pathway is an important component of normal development, its aberrant activation leads to a more aggressive phenotypes, suggesting it can be targeted for the treatment of pancreatic cancer. Since riluzole inhibited Wnt/β-catenin/TCF-LEF1 pathway, we next sought to examine the effects of riluzole on downstream targets of Wnt pathway. These downstream targets regulate cell cycle, cell growth, components of Wnt pathway, and metabolism. Riluzole inhibited the expression of cyclin D1 in both MIA PaCa-2 and AsPC-1 cells (Fig. 4A). Riluzole treatment of MIA PaCa-2 cells resulted in an inhibition of G1 stage and an increase in G2/M stage of cell cycle (Fig. 4B). Riluzole treatment also caused a slight but significant increase in S phase of cell cycle in MIA PaCa-2 cells. By comparison, 10 μM dose of riluzole has no significant effect on cell cycle in AsPC-1 cells (Fig. 4B). Higher doses of riluzole (20 and 40 μM) inhibited G1 stage and increased G2/M stage of cell cycle. A slight inhibition in S stage was seen with the highest dose (40 μM) of riluzole in AsPC-1 cells. Transcription factor TCF/LEF induces the expression of cMyc, Axin1, GSK3B, pyruvate dehydrogenase kinase 1 (PDK1), and monocarboxylate lactate transporter 1 (MCT-1). Riluzole inhibited the expression of cMyc, Axin1, GSK3B, PDK1, and MCT1 in both MIA PaCa-2 and AsPC-1 cells (Fig. 4C,D). These data suggest that riluzole can regulate cell cycle, cell proliferation, and glucose metabolism in pancreatic cancer cells by modulating the expression of components of Wnt pathway and its down-stream targets.
Riluzole inhibited Bcl-2 and induced Bax expression and disrupted mitochondrial membrane potential in pancreatic cancer cells. Bcl-2 family members play a major role in regulating mitochondrial functions including mitochondrial membrane potential. We, therefore, measured the expression of antiapoptotic Bcl-2, proapoptotic Bax and mitochondrial membrane potential. Riluzole inhibited Bcl-2 and induced Bax expression in pancreatic cancer cells (Fig. 5A-D). Since Bcl-2 and Bax act at the level of mitochondria and regulate permeability transition, we measured the effects of riluzole on mitochondrial membrane potential. Treatment of AsPC-1 and MIA PaCa-2 cells with riluzole caused a drop in mitochondrial membrane potential in
Glutamine is required for pancreatic cancer. Pancreatic cancer cells are addicted to amino acid glutamine to fuel anabolic processes 34,52 . In order to test the requirement of the glutamine for PDAC, we have grown PANC-1 cells in the glutamine free medium and also in the presence of glutaminase (GLS) inhibitors [(bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulphide (BPTES) or 6-diazo-5-oxo-L-norleucin (DON)]. The data demonstrate that inhibition of GLS by BPTES or DON induced apoptosis in PANC-1 cells (Fig. 7A,B). Similarly, glutamine deprivation induced apoptosis in PANC1 cells. GLS inhibitors induced apoptosis to the same extent as glutamine deprivation. These data suggest that glutamine is required for PDAC.
Riluzole inhibited glutamate release, and glutathione (GSH) level, and elevated reactive oxygen species (ROS) in pancreatic cancer cells and CSCs. Since riluzole regulates glutamate metabo-   (Fig. 7C). Since glutathione (GSH) participates in oxidative stress response and defends against cellular toxicity, we sought to examine the effects of riluzole on intracellular GSH and ROS production. Riluzole inhibited intracellular GSH level in pancreatic cancer cells and CSCs (Fig. 7D). Treatment of AsPC-1 and PANC-1 cells with riluzole resulted in elevation of ROS reaching a plateau at 150 and 120 min, respectively (Fig. 7E,F). Pre-treatment of cells with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited riluzole-induced ROS production ( Fig. 7E-G). These data suggest that riluzole can increase intracellular glutamate, inhibit GSH and elevate ROS, which may be one of the mechanisms of apoptosis induction through mitochondrial dysfunction. to test the efficacy of new drugs for the treatment of pancreatic cancer 44,54 . Since riluzole inhibited cell proliferation and colony formation, and induced apoptosis in vitro, we next sought to examine the effects of riluzole on pancreatic cancer growth and development in KPC (Pdx1-Cre, LSL-Trp53 R172H , and LSL-Kras G12D ) mice by treating them for three months. Treatment of KPC mice with riluzole inhibited pancreas weight, which was similar to that of untreated Cre mice (Fig. 8A). Control group of KPC mice developed PanIN 1-3 lesions and PDAC at about 4.5 months (Fig. 8B). By comparison, numbers of PanIN 1 and 2 lesions were significantly inhibited in the riluzole-treated group. Interesting, PanIN3 and PDAC were not observed in the riluzole-treated group. Riluzole inhibited pancreatic cancer growth and development in KPC mice. Overall, our transgenic mice data suggest that riluzole is effective in inhibiting pancreatic cancer growth and development in mice, and can be used for the treatment of pancreatic cancer.

Discussion
We have shown for the first time that riluzole inhibits pancreatic cancer growth and development by regulating glucose and glutamine metabolisms. The existence of cancer stem cells (CSCs) in the pancreas hinders the development of new drugs because CSCs can contribute towards therapy failure and drug resistance. Riluzole not only inhibits growth of cancer cells but also cancer stem cells which are generally responsible for drug resistance and chemotherapy failure. Furthermore, riluzole affects cancer cell metabolisms and mitochondrial homeostasis which can modify responses to therapy. Since Wnt/β-catenin/TCF-LEF pathway is highly activated in pancreatic www.nature.com/scientificreports/ cancer, inhibition of Wnt/β-catenin/TCF-LEF pathway by riluzole will not only inhibit cancer cell proliferation but also regulate those genes which play major roles in cancer cell metabolisms.
In the present study, anticancer activities of riluzole were observed in pancreatic cancer cells which show genetic variability in Kras and p53 status. Riluzole was also effective in pancreatic CSCs which generally do not respond to anticancer drugs and are responsible for drug resistance and cancer relapse. In addition to apoptosis, riluzole treatment also caused growth arrest at G2/M stage and reduced G1 stage of cell cycle which was accompanied by inhibition of cyclin D1 expression. Similarly, another study has demonstrated that riluzole inhibited cell viability and colony formation, blocked cell cycle, and induced apoptosis in pancreatic cancer cells 55 . Furthermore, in other studies riluzole exerted anti-tumor activities in breast cancer cells independent of metabotropic glutamate receptor-1, and inducing mitotic arrest 56,57 . Interestingly, riluzole was not effective in human normal pancreatic ductal epithelial cells, suggesting its activity was limited to malignant cells and thus offers hope for the treatment of pancreatic cancer.
Mitochondria glutamine metabolism plays an essential role in maintaining mitochondrial functions and regulates cellular sensitivity to DNA damage 11 . Glutamine oxidizes in mitochondria and produces ATP. Pancreatic cancer cells are addicted to glutamate for survival [58][59][60] . Riluzole inhibits glutamate release through inactivation of voltage-dependent ion channels [19][20][21] . In prostate cancer, serum glutamate levels directly correlate with Gleason score and glutamate blockade decreases proliferation, migration, and invasion and induces cell death 61 . Blocking glutamate release by riluzole inhibits cell proliferation in glioblastoma, melanoma, breast and prostate cancer 19,56,[62][63][64] . Since riluzole inhibited glutamate release and GSH level, and increased ROS production in pancreatic cancer cells and CSCs, this could be considered as one of the mechanisms of apoptosis induction.
Upregulation of WNT/β-catenin pathway induces aerobic glycolysis (known as Warburg effect), through activation of GLUT, PDK1, pyruvate kinase M2 (PKM2), MCT-1, LDH-A and inactivation of pyruvate dehydrogenase complex. Oncogenic Myc regulates the expression of glycolysis genes, such as PDK1, GLUT1, HK2, and LDH-A 65,66 . WNT/β-catenin pathway directly regulates Myc expression. The aerobic glycolysis supplies a large part of glucose into lactate regardless of oxygen. Aerobic glycolysis is less efficient in producing ATP compared to oxidative phosphorylation. Phosphorylation of PDK-1 inhibits the PDH, and a large part of pyruvate cannot be converted into acetyl-CoA in mitochondria and only a part of acetyl-CoA can enter the TCA cycle. Cytosolic pyruvate is converted into lactate through the enzymatic activity of LDH-A. In the present study, riluzole inhibited c-Myc and GLUT-2 expression, glucose uptake, LDH-A expression, and NAD + levels. Furthermore, knockdown of β-catenin results in reduced glutamate transporter (GLT-1) and glutamine synthetase (GS) expression in astrocytes 35 . Similar to the action of riluzole, inhibition of mitochondrial ATP production downregulated Wnt/beta-catenin signaling pathway 67,68 .
Bcl-2 family members regulate cell growth, survival, and apoptosis [69][70][71][72] . Mainly, anti-apoptotic members such as Bcl-2 and Bcl-X L , enhance cell growth and proliferation, whereas pro-apoptotic members such as Bax and Bad, induce apoptosis. Following cellular stress, Bak and/or Bax are activated and compromise the integrity of the outer mitochondrial membrane (OMM) resulting in permeabilization of mitochondrial outer membrane. As a result of MOMP, pro-apoptotic proteins (e.g., cytochrome c) move to the cytoplasm where they activate In the present study, riluzole inhibited the expression of Bcl-2 and induced the expression of Bax and caused a drop in mitochondrial membrane potential leading to induction of apoptosis. These data suggest that riluzole can act at the level of mitochondrial to regulate apoptosis.
In conclusion, the proapoptotic and antiproliferative effects of riluzole are exerted through rewiring of mitochondrial signals that are specific to pancreatic cancer cells. Glutamine, a mitochondrial substrate, could be required for maintenance of mitochondrial membrane potential and integrity and for support of the NADPH production needed for redox control and macromolecular synthesis. Furthermore, metabolic reprogramming of pancreatic cancer mediated by glutamate inhibition elicits unique vulnerabilities to malignant cells, but not to normal pancreatic epithelial cells. Our in vitro and in vivo data suggest that riluzole can be used for the treatment of pancreatic cancer. CSCs were treated with riluzole (0-10 µM) for 24 h. Intracellular total GSH was detected by measuring the product of glutathionylated DTNB at 405 nm (GSH assay kit, Cayman Chemical). Data represent mean ± SD (n = 4). * = significantly different from control, P < 0.05. (E and F), AsPC-1 and PANC-1 cells were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0-10 µM) for 24 h. Cells were labelled with 2' ,7'-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured for various time points (0-360 min) with a fluorometer using excitation at 495 nm and emission at 529 nm (Cellular Reactive Oxygen Species Detection Assay Kit, Abcam). Data represent mean ± SD (n = 4). *, @, #, $. %, &, and ** = significantly different from control; p < 0.05. (G), AsPC-1 and Pan CSCs were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0-10 µM) for 24 h. Cells were labelled with 2' ,7'-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured at 120 min with a fluorometer using excitation at 495 nm and emission at 529 nm. Data represent mean ± SD (n = 4). * = significantly different from control and each other; p < 0.05.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.  License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.